The invention relates to a novel process for the preparation of (1R,4S)- or (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene of the formulae 
and/or of (1S,4R)- or (1R,4S)-amino alcohol derivatives of the general formulae 
and to novel microorganisms which are able to utilize a cyclopentene derivative of the general formula 
as sole nitrogen source, as sole carbon source or as sole carbon and nitrogen source.
The invention further relates to enzyme extracts and enzymes having N-acetylamino-alcohol hydrolase activity obtainable from these microorganisms.
(1R,4S)-1-Amino-4-(hydroxymethyl)-2-cyclopentene of the formula I is an important intermediate for the preparation of carbocyclic nucleosides such as, for example, Carbovir(copyright) (Campbell et al., J. Org. Chem. 1995, 60, 4602-4616).
Processes for the preparation of (1R,4S)-amino-4-(hydroxymethyl)-2-cyclopentene are described by Campbell et al. (ibid) and by Park K. H. and Rapoport H. (J. Org. Chem. 1994, 59, 394-399).
The precursor used in these processes is either D-glucono-xcex4-lactone or D-serine, and about 15 synthesis stages are necessary to form (1R,4S)-N-tert-butoxycarbonyl-4-hydroxymethyl-2-cyclopentene, which is then deprotected to give (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene. These two processes are costly, elaborate and cannot be implemented industrially.
WO 93/17020 describes a process for the preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene, wherein (1R,4S)-4-amino-2-cyclopentene-1-carboxylic acid is reduced with lithium aluminium hydride to the desired product.
The disadvantage of this process is, on the one hand, that the double bond of the cyclopentene ring is also reduced, the lithium aluminium hydride is difficult to handle, and, on the other hand, that it is too costly.
Taylor, S. J. et al. (Tetrahedron: Asymmetry Vol. 4, No. 6, 1993, 1117-1128) describe a process for the preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene starting from (xc2x1)-2-azabicyclo [2.2.1]hept-5-en-3-one as precursor. In this case, the precursor is converted by means of microorganisms of the species Pseudomonas solanacearum or Pseudomonas fluorescens into (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one, which is then converted with di-tert-butyl dicarbonate into (1R,4S)-N-tert-butoxycarbonyl-2-azabicyclo [2.2.1]hept-5-en-3-one, which is reduced with sodium borohydride and trifluoroacetic acid to the desired product. This process is much too costly.
In addition, Martinez et al. (J. Org. Chem. 1996, 61, 7963-7966) describe a 10-stage synthesis of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene starting from diethyl dialkylmalonate. This process also has the disadvantage that it is elaborate and cannot be implemented industrially.
It was an object of the present invention to provide a simple process for the preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene.
This object is achieved with the microorganisms of the invention according to claim 1, and enzyme extracts therefrom, with the enzymes of the invention according to claim 4 and with the process of the invention according to claim 7.
The microorganisms of the invention can be isolated from soil samples, sludge or wastewater with the assistance of conventional microbiological techniques.
The microorganisms are isolated according to the invention by cultivating them in a nutrient medium containing one or more cyclopentene derivatives of the general formula 
in which R1 denotes C1-C4-alkyl, C1-C4-alkoxy, aryl or aryloxy,
as sole carbon and nitrogen source
as sole nitrogen source with a suitable carbon source or
as sole carbon source with a suitable nitrogen source,
in a conventional way.
It is possible to use as C1-C4-alkyl for example methyl, ethyl, propyl, isopropyl or butyl. It is possible to use as C1-C4-alkoxy for example methoxy, ethoxy, propoxy, isopropoxy, butoxy or tert-butoxy. It is possible to use as aryl for example phenyl or benzyl. Benzyl is preferably used. It is possible to use as aryloxy for example benzyloxy or phenoxy. Accordingly, the following examples are suitable as cyclopentene derivative of the general formula VII:
1-acetylamino-4-hydroxymethyl-2-cyclopentene, 1-butyrylamino-4-hydroxymethyl-2-cyclopentene or 1-phenylacetylamino-4-hydroxymethyl-2-cyclopentene.
It is expedient to select from the culture obtained by cultivation those which utilize the (1R,4S) isomer of the cyclopentene derivative of the formula VII as sole nitrogen source, as sole carbon source or as sole carbon and nitrogen source.
The microorganisms can use as suitable nitrogen source, for example, ammonium, nitrates, amino acids or ureas as substrate for growth. The microorganisms can use as suitable carbon source, for example, sugars, sugar alcohols, C2-C4-carboxylic acids or amino acids as substrate for growth. Hexoses such as glucose or pentoses can be used as sugars. Glycerol, for example, can be used as sugar alcohol. Acetic acid or propionic acid can be used, for example, as C2-C4-carboxylic acids. Leucine, alanine, asparagine can be used, for example, as amino acids.
The selection medium and culture medium which can be used are those conventional among those skilled in the art, such as, for example, the one described in Table 1 or a complete medium (medium containing yeast extract), preferably using the one described in Table 1.
During the culturing and selection, the active enzymes of the microorganisms are expediently induced. The cyclopentene derivatives of the general formula VII can be used as enzyme inducer.
The culturing and selection normally takes place at a temperature from 20xc2x0 C. to 40xc2x0 C., preferably from 30xc2x0 C. to 38xc2x0 C. and at a pH between 5.5 and 8.0, preferably between 6.8 and 7.8.
Preferred microorganisms are those of the genus Rhodococcus, Gordona, Arthrobacter, Alcaligenes, Agrobacterium/Rhizobium, Bacillus, Pseudomonas or Alcaligenes/Bordetella, in particular of the species Rhodococcus erythropolis CB 101 (DSM 10686), Alcaligenes/Bordetella FB 188 (DSM 11172), Arthrobacter sp. HSZ 5 (DSM 10328), Rhodococcus sp. FB 387 (DSM 11291), Alcaligenes xylosoxydans ssp. denitrificans HSZ 17 (DSM 10329), Agrobacterium/Rhizobium HSZ 30, Bacillus simplex K2, Pseudomonas putida K32, or Gordona sp. CB 100 (DSM 10687) and their functionally equivalents variants and mutants. Deposition in accordance with the Budapest Treaty at the Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH (DSMZ), Mascheroderweg 1b, D-38124 Braunschweig, took place on 20.05.1996 for the microorganisms DSM 10686 and 10687, on 6.11.1995 for the microorganisms DSM 10328 and DSM 10329, on 8.10.1996 for the microorganism DSM 11291 and on 20.09.1996 for the microorganism DSM 11172.
xe2x80x9cFunctionally equivalent variants and mutantsxe2x80x9d mean microorganisms having essentially the same properties and functions as the original microorganisms. Variants and mutants of this type can be produced by chance, for example by UV radiation.
Taxonomic Description of Rhodococcus erythropolis CB 101 (DSM 106 86)
1. Morphology and color of the colonies: short branched hyphae which, when old, disintegrate into rods and cocci, colonies glistening and partly confluent, beige with pink tinge, RAL 1001;
2. Diagnosed amino acid of the peptidoglycan: mesodiaminopimelic acid;
3. Mycolic acids: Rhodococcus mycolic acids; determination of the mycolic acid chain length (C32-C44) and comparison of the data with the entries in the DSM mycolic acid data bank revealed very great similarity with the patterns of the Rhodococcus erythropolis strains (similarity 0.588).
4. Fatty acid pattern: unbranched, saturated and unsaturated fatty acids plus tuberculostearic acid.
5. On partial sequencing of the 16S rDNA of the strain, a high level of agreement (100%) was found with the sequences of the specific regions of Rhodococcus erythropolis. 
The identification result is unambiguous because three mutually independent methods (mycolic acids, fatty acids, 16S rDNA) have assigned the strain to the species Rhodococcus erythropolis. 
Taxonomic Description of Gordona sp. CB 100 (DSM 10687)
1. Morphology and color of the colonies: short branched hyphae which, when old, disintegrate into rods and cocci, colonies pale orange, (RAL 2008);
2. Diagnosed amino acid of the peptidoglycan: mesodiaminopimelic acid;
3. Menaquinone pattern: MK-9 (H2) 100%;
4. Mycolic acids: Gordona mycolic acids; the mycolic acid chain length (C50C60) was determined by high temperature gas chromatography. This pattern corresponds to the pattern found in representatives of the genus Gordona.
5. Fatty acid pattern: unbranched, saturated and unsaturated fatty acids plus tuberculostearic acid.
6. On partial sequencing of the 16S rDNA of the strain, only a relatively low agreement of 98.8% could be found with the sequences of the specific regions of Gordona rubropertincta. 
On the basis of the available results (menaquinones, mycolic acids, fatty acids, 16S rDNA), although the isolate can be unambiguously assigned to the genus Gordona it is not possible on the basis of the results to make an assignment to a known Gordona species. It is therefore to be assumed that the strain DSM 10687 is a new and previously undescribed species of the genus Gordona.
Partial sequencing of the 16S rDNA revealed comparably large similarities of about 96% with representatives of the genera Agrobacterium and Rhizobium. Unambiguous assignment to a species described within these genera is not possible.
Analysis of the cellular fatty acids yielded confirmation of the assignment to the genus Bacillus.
Partial sequencing of the 16S rDNA revealed a similarity of 100% with Bacillus simplex.
The profile of cellular fatty acids is typical of Pseudomonas putida. 
Partial sequencing of the 16S rDNA revealed similarities of about 98% with Pseudomonas mendocina and Pseudomonas alcaligenes. The similarity with Pseudomonas putida was 97.4%.
Taxonomic Description of Rhodococcus sp. FB 387 (DSM 11291)
1. Morphology and colour of the colonies: short branched hyphae which, when old, disintegrate to rods and cocci, colonies matt, pale red-orange RAL 2008;
2. Diagnosed amino acid of the peptidoglycan: mesodiaminopimelic acid;
3. Mycolic acids: Rhodococcus mycolic acids;
Determination of the mycolic acid chain length (C32-C44) and comparison of the data with the entries in the DSMZ mycolic acid data bank revealed only very small similarity with the patterns of Rhodococcus ruber strains (similarity 0.019). This correlation factor is too low to be used for species identification.
4. Fatty acid pattern: unbranched, saturated and unsaturated fatty acids plus tuberculostearic acid.
xe2x80x83This fatty acid pattern is diagnostic for all representatives of the genus Rhodococcus and its close relatives such as Mycobacterium, Nocardia and Gordona. An attempt was made by including the qualitative and quantitative differences in the fatty acid pattern to carry out a differentiation to the species level. Numerical methods were used to compare the fatty acid pattern of Rhodococcus sp. FB 387 with the entries in the data bank. It was not possible with this method either to assign Rhodococcus sp. FB 387, because of the small similarity (0.063), to any described Rhodococcus species.
5. On partial sequencing of the 16S rDNA of the strain, 96-818 was assigned to Rhodococcus opacus with a correlation of 97.9%. This sequence agreement is far below that of 99.5% required for unambiguous species assignment in this taxon.
On the basis of the available results, it can be assumed that the strain Rhodococcus sp. FB 387 is a new and not previously described Rhodococcus species.
The enzymes of the invention, the N-acetylamino-alcohol hydrolases which are able to hydrolyse cyclopentene derivatives of the above formula VII, can be obtained, for example, by disruption of the microorganism cells of the invention in a way conventional for the skilled person. It is possible to use for this for example the ultrasound or French press method. These enzymes can be obtained for example from Rhodococcus erythropolis CB 101 (DSM 10686) microorganisms. Enzymes obtainable from the microorganisms of the invention, especially Rhodococcus erythropolis CB 101 (DSM 10686), preferably have the following properties:
a) a pH optimum of pH 7.0xc2x11.0;
b) a temperature optimum between 25xc2x0 and 30xc2x0 C. at a pH of 7.0; and
c) a KM for the substrate 1-acetylamino-hydroxymethyl-2-cyclopentene of 22.5 mMxc2x17.5 mM (30xc2x0 C., 100 mM phosphate buffer, pH 7.0).
Sequence analysis of an enzyme obtainable from Rhodococcus erythropolis CB 101 (DSM 10686) further revealed:
d) an N-terminal amino acid sequence of Thr-Glu-Gln-Asn-Leu-His-Trp-Leu-Ser-Ala-Thr-Glu-Met-Ala-Ala-Ser-Val-Ala-Ser-Asn;
and a molecular weight determination revealed:
e) a molecular weight of 50 kD determined by SDS-PAGE.
Enzymes like those obtainable from the microorganisms of the invention, for example Rhodococcus erythropolis CB 101 (DSM 10686), hydrolyse, for example, in particular 1-acetylamino-4-hydroxymethyl-2-cyclopentene, 1-butyrylamino-4-hydroxymethyl-2-cyclopentene, 1-propionylamino-4-hydroxymethyl-2-cyclopentene and 1-isobutyrylamino-4-hydroxymethyl-2-cyclopentene.
The process of the invention for the preparation of (1R,4S)- or (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene of the formulae 
and/or of (1S,4R)- or (1R,4S)-amino alcohol derivatives of the general formulae 
in which R1 has the stated meaning, can be carried out for example by, in a first stage, acylating (xc2x1)-2-azabicyclo[2.2.1]hept-5-en-3-one of the formula 
to give a (xc2x1)-2-azabicyclo[2.2.1]hept-5-en-3-one derivative of the general formula 
in which R1 has the stated meaning.
The precursor (xc2x1)-2-azabicyclo[2.2.1]hept-5-en-3-one can be prepared as disclosed in EP-B 0 508 352.
The acylation can be carried out with a carbonyl halide of the general formula 
in which X denotes a halogen atom, and R1 has the stated meaning, or with a carboxylic anhydride of the general formula 
in which R1 has the stated meaning.
F, Cl, Br or I can be used as halogen atom X. Cl or F is preferably used.
Examples of carbonyl halides are: acetyl chloride, chloroacetyl chloride, butyryl chloride, isobutyryl chloride, phenylacetyl chloride, benzyl chloroformate (Cbz-Cl), propionyl chloride, benzoyl chloride, allyl chloroformate or tert-butyl fluoroformate. Examples of carboxylic anhydrides are: di-tert-butyl dicarbonate, butyric anhydride, acetic anhydride or propionic anhydride.
The acylation can be carried out without solvent or with an aprotic solvent.
The acylation is expediently carried out in an aprotic solvent. Examples of suitable aprotic solvents are pyridine, acetonitrile, dimethylformamide, tetrahydrofuran, toluene, methylene chloride, N-methylpyrrolidone or mixtures thereof. The solvent preferably used is pyridine or acetonitrile, in particular a mixture of pyridine and acetonitrile.
The acylation is expediently carried out at a temperature from xe2x88x9280 to 50xc2x0 C., preferably from 0 to 25xc2x0 C.
In a second stage of the process, the (xc2x1)-2-azabicyclo[2.2.1]hept-5-en-3-one derivative of the formula VI can be reduced to give a cyclopentene derivative of the general formula 
in which R1 has the stated meaning.
The reduction is expediently carried out with an alkali metal borohydride or alkaline earth metal borohydride, with an alkali metal aluminium hydride or alkaline earth metal aluminium hydride or with Vitride (sodium bis (2-methoxyethoxy) aluminium hydride). Sodium or potassium aluminium hydride can be used as alkali metal aluminium hydride. Sodium or potassium borohydride can be used as alkali metal borohydride. Calcium borohydride can be used as alkaline earth metal borohydride.
The reduction is expediently carried out in a protic solvent. Protic solvents which can be used are lower aliphatic alcohols such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, sec-butanol, tert-butanol, or water, or mixtures thereof.
The reduction is expediently carried out at a temperature from xe2x88x9240 to 40xc2x0 C., preferably from 0 to 20xc2x0 C.
The conversion of the cyclopentene derivative of the general formula VII into the (1R,4S)- or (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene of the formulae 
is carried out according to the invention either by means of microorganisms or enzyme extracts therefrom, by means of penicillin G acylases or by means of enzymes having N-acetylamino-alcohol hydrolase activity. This biotransformation results not only in the (1R,4S)- or (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene of formula I or II, which is isolated where appropriate, but also in the (1S,4R)- or (1R,4S)-amino alcohol derivative of the general formulae 
in which R1 has the stated meaning. The latter can likewise be isolated where appropriate.
All microorganisms which utilize a cyclopentene derivative of the general formula VII as sole nitrogen source, as sole carbon source or as sole carbon and nitrogen source are suitable. The biotransformation is expediently carried out with microorganisms which utilize the (1R,4S) isomer of the cyclopentene derivative as sole carbon source, as sole carbon and nitrogen source or as sole nitrogen source.
The biotransformation is preferably carried out by means of microorganisms of the genus Alcaligenes/Bordetella, Rhodococcus, Arthrobacter, Alcaligenes, Agrobacterium/Rhizobium, Bacillus, Pseudomonas or Gordona, in particular of the species Algaligenes/Bordetella FB 188 (DSM 11172), Rhodococcus erythropolis CB 101 (DSM 10686), Arthrobacter sp. HSZ 5 (DSM 10328), Rhodococcus sp FP 387 (DSM 11291), Alcaligenes xylosoxydans ssp. denitrificans HSZ 17 (DSM 10329), Agrobacterium/Rhizobium HSZ 30, Bacillus simplex K2, Pseudomonas putida K32, or Gordona sp. (DSM 19687), and with the functional equivalent variants and mutants thereof. These microorganisms are, as already described, deposited in accordance with the Budapest Treaty.
The microorganism species very particularly suitable for the process are Alcaligenes/Bordetella FB 188 (DSM 11172), Rhodococcus erythropolis CB 101 (DSM 10686) and Gordona sp. CB 100 (DSM 10687).
The biotransformation can be carried out, after conventional initial cultivation of these microorganisms, with quiescent cells (non-growing cells no longer requiring a carbon and energy source) or with growing cells. The biotransformation is preferably carried out with quiescent cells.
The enzymes according to the invention which are suitable for the process, the N-acetylamino-alcohol hydrolases, can be obtained by the methods described above and have the properties already described above.
Suitable penicillin G acylases are obtained from many microorganisms such as, for example, bacteria or actinomycetes, specifically from the following microorganisms: Escherichia coli ATCC 9637, Bacillus megaterium, Streptomyces lavendulae ATCC 13664, Nocardia sp. ATCC 13635, Providencia rettgeri ATCC 9918, Arthrobacter viscosus ATCC 15294, Rhodococcus fascians ATCC 12975, Streptomyces phaeochromogenes ATCC 21289, Achromobacter ATCC 23584 and Micrococcus roseus ATCC 416. Penicillin G acylases which can be bought are used in particular, such as penicillin G acylase EC 3.5.1.11 from E. coli (Boehringer Mannheim) or from Bacillus megaterium. 
Immobilized penicillin G acylases are used in a preferred embodiment.
The biotransformation can be carried out in media usual in the art, such as, for example, in low-molarity phosphate, citrate or Hepes buffer, in water, in complete media such as, for example, Nutrient Yeast Broth (NYB) or in that described in the table. The biotransformation is preferably carried out in the medium shown in Table 1 or in low-molarity phosphate buffer.
The biotransformation is expediently carried out with a single or continuous addition of the cyclopentene derivative (formula VII) so that the concentration does not exceed 10% by weight, preferably 2% by weight.
The pH during the biotransformation can be in a range from 5 to 9, preferably from 6 to 8. The biotransformation is expediently carried out at a temperature from 20 to 40xc2x0 C., preferably from 25 to 30xc2x0 C.
If the (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene is formed during the biotransformation, this can be converted into the (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene by acid hydrolysis, for example with hydrochloric acid.